The new direction taken in this competitive renewal comes from our longstanding interest in the signaling mechanisms of the pleiotropic cytokine TNF. While TNF-induced NF-?B activation and apoptosis are known for years, recent elucidation of TNF-induced programmed necrosis and the switching mechanisms among these three alternative cell fates have revolutionized how we look at this classical cytokine. In a simplified overview, RIP1 is at the center of this cell fate regulation. When RIP1 is ubiquitinated in the TNF signaling complex, NF-?B activation ensues, resulting in cell survival, proliferation and differentiation. When RIP1 ubiquitination is blocked by removal of the E3 ligases cIAP1 and cIAP2 through genetic ablation, RNAi knock down, or IAP antagonists, RIP1 forms a secondary complex with FADD and caspase-8 to initiate apoptotic cell death. If apoptosis is inhibited by caspase inhibitors or under certain physiological conditions, RIP1 associates with RIP3 to form the necrosome to initiate programmed necrosis or necropolis. Whereas necrosis was originally thought to be associated with non-specific cellular damage, recent evidence clearly showed that necrosis is critical for embryonic development, host defense and other biological processes. Despite the biological importance of the necrosome in the signal transduction of TNF and other death receptors, no structural and mechanistic information is available. In this application, we propose to assemble the RIP1/RIP3 necrosome and related complexes and to elucidate the molecular basis of the signal transduction. PUBLIC HEALTH RELEVANCE: Engagement of the necrotic pathway in appropriate physiological contexts is important for embryonic development and initiates protective immune responses. However, the complexity of this pathway also renders itself susceptible to interruption and dysregulation, leading to its association with many human diseases, such as susceptibility to viral infection and unwanted cell death in ischemic injury in heart attack and stroke. Understanding this pathway at the molecular level would contribute to both health and disease.